Permeation layer attachment chemistry and method

ABSTRACT

Electronically addressable microchips having covalently bound permeation layers and methods of making such covalently bonded permeation layers to microchips are provided. The covalent bonding is derived from combining the use of electrodes with silane derivatives. Such chemistry provides the ability to apply an electronic bias to the electrodes of the microchip while preventing permeation layer delaminating from the electrode surface.

FIELD OF THE INVENTION

[0001] This invention relates to the attachment of a layer of polymericmaterial to a substrate surface. More particularly, this inventionrelates to chemistries and methods for covalently attaching a porouspolymeric material to an electrically conductive substrate, such as ametal electrode of a microchip circuit.

BACKGROUND OF THE INVENTION

[0002] The following description provides a summary of informationrelevant to the present invention. It is not an admission that any ofthe information provided herein is prior art to the presently claimedinvention, nor that any of the publications specifically or implicitlyreferenced are prior art to the invention.

[0003] In the art of electronically addressable microchips that are usedto direct biomaterials, such as nucleic acids and proteins, from onepoint in a solution to another, the microchips should be designed sothat electric potential from the microchip electrodes will translate tothe solution overlying the microchip such that any electrochemistryoccurring from the electrode surface will neither damage the electrodesthemselves, nor any biomaterials in the solution. Generally, protectionfrom such damage is provided by the use of a porous membrane layerdeposited over the microchip electrodes. Usually, such layer comprisesmaterials derived from natural or synthetic polymers such as agarose orpolyacrylamide, respectively. These types of materials allowelectrochemical products generated at the electrode surface to travelthrough their porous matrix or ‘permeation layer’ and into the solutionimmediately above the electrodes.

[0004] Although materials such as those noted above have been founduseful in the role of a porous membrane having desired qualities, it hasbeen found that because of the methodologies commonly used to layer suchmembranes onto the microchip substrate, the membranes are prone toseparate or ‘delaminate’ from the electrode surface. It is believed thisdelamination is caused by a change in the chemical make-up at theinterface between the permeation layer and the electrode resulting fromthe application of electronic potential at the electrode and by physicaldisruption from charged ions and gases emanating from the electrode.Such delamination can be viewed from the standpoint of‘microdelamination’ and ‘macrodelamination’.

[0005] Microdelamination involves the electrochemical degradation of thechemical interface between the permeation layer and the electrodeitself. It is observed by the formation of raised bulges in thepermeation layer, or by ringlets visible due to defraction of light fromthe delaminated layer when appropriately viewed by a confocal microscopeand results in the loss of consistency in permeation layer performance(possibly due to the loss of control over the electric fielduniformity). Macrodelamination, on the other hand, is caused by amismatch of the surface energies between the permeation layer and thechip substrate and results in permeation layer peeling (lift-off) whichcan extend across the entire microchip surface. Since the permeationlayer provides a means for chemical anchorage of analytes present in theliquid overlay, its physical loss by macrodelamination results incatastrophic chip failure during bioassays.

[0006] Electronically addressable systems such as the microchipsconsidered herein follow Ohm's law which establishes the relationshipbetween the voltage drop (V) between two electrodes (i.e., the anode,placed at a positive potential and the other, the cathode, placed at anegative potential), and the electric current (I) which flows betweenthese electrodes, as follows:

V=R×I  (1)

[0007] where R is the electrical resistance of the medium between theanode and the cathode. In systems where a permeation layer is presentover such electrodes, the value of R is greatly determined by thephysical and chemical nature of said permeation layer. Thus, accordingto formula (1), the difference between the electronic potentials appliedto the electrodes is directly proportional to the intensity or densityof the electric current which flows through them. The inventiondescribed in this Letters Patent uses a relationship between electriccurrent and voltage wherein electric current densities are at least 0.04nA/μm² and/or voltage drops are between 1 and 3 V. The electric currentdensity is defined as the electric current divided by the area of theelectrode used to support it.

[0008] Additionally, the effectiveness of the translocation of chargedbiomolecules such as nucleotide oligomers within anelectronically-driven system such as that described herein depends onthe generation of the proper gradient of positively and negativelycharged electrochemical species by the anode and cathode, respectively.For example, effective nucleic acid (i.e. either DNA or RNA) transportmay be accomplished by generation of protons and hydroxyl anions whenthe potential at the anode is greater than +1.29 V with respect to a‘saturated calomel electrode’ (SCE). When subjected to such demandingoperating conditions, noncovalently-attached permeation layers prove tobe unsatisfactory since such systems are likely to experience micro- andsometimes macrodelamination. Moreover, the transport efficiency ofcharged molecules increases with increasing current density, thusdriving the desire for operation at higher voltage drops and currentdensities and, thus, the need for evermore robust permeation layers.

[0009] Therefore, a need still remains for methodologies for keepingpermeation layers from delaminating from electronic microchip substratesand particularly from the electrode pads themselves. We have discoveredan improvement in permeation layer attachment chemistry that provides asignificant increase in permeation layer performance. Specifically, wehave solved the problem of micro- and macrodelamination by discovery ofa covalent chemistry linkage system that, as applied to electronicallyaddressable microchip art, can be incorporated between the microchip andthe permeation layer matrix. This chemistry is applicable to a varietyof permeation layer compositions, including polymers, hydrogels,glyoxylagarose, polyacrylamide, polymers of methacrylamide, materialsmade from other synthetic monomers, and porous inorganic oxides createdthrough a sol-gel process, and is able to withstand current densities ofat least 0.04 nA/μm² and/or voltage drops between 1 and 3 V.

SUMMARY OF THE INVENTION

[0010] The current invention provides a unique system for the covalentattachment of a porous ‘permeation layer’ to the surface ofelectronically addressable microchips. In a preferred embodiment, thecovalent attachment is between chemical moieties of the permeation layerand metal/silicide, metal/metal, or organic electrodes. Preferredmetal/silicide electrodes include platinum silicide (PtSi), tungstensilicide (WTi), titanium silicide (TiSi), and gold silicide (AuSi).Preferred metal/metal electrodes include platinum/titanium (PtTi) andgold/titanium (AuTi). Preferred organic electrodes include materialssuch as poly(phenylene vinylene), polythiophene, and polyaniline.

[0011] In an example of this embodiment, the covalent attachmentcomprises a linking moiety that provides an attachment mechanism forbonding the linker to the silanol moiety of a metal/Si surface and aseparate moiety for bonding the linker to the permeation layer. Wheremetal/metal and organic electrodes are employed, the attachmentmechanism of the linker to the electrode is the same in that the moietyof the linker attaching to the electrode will react with specific metalsand reactive centers on organic molecules to form covalent bonds.

[0012] In a particularly preferred embodiment, the linking moiety isdefined by the formula:

[0013] where X=acrylate, methacrylate, acrylamide, methacrylamide,allyl, vinyl, acetyl, amine (substituted or not), epoxy or thiol;

[0014] SPACER=alkyl, aryl, mono- or polyalkoxy (such as ethyleneglycolor polyethyleneglycol), mono- or polyalkylamine, mono- or polyamide,thioether derivatives, or mono- or polydisulfides;

[0015] A and B=any combination of Oxygen-R, where R=H, alkyl such asmethyl, ethyl, propyl, isopropyl or other linear or branchedhydrocarbon, Cl, Br or a moiety functionality similar to that ofX-SPACER; and

[0016] C=Oxygen-R, where R=H, alkyl such as methyl, ethyl, propyl,isopropyl or other linear or branched hydrocarbon, Cl, Br, or any otherhydrolyzable moiety.

[0017] In the example of the metal/Si electrodes, these linkage groups,which contain a suicide group can react with hydroxyl groups bonded toan oxygen moiety of the electrode surface. On the other end of thelinker, the X moiety comprises chemical groups that are available tocovalently react with reactive centers of the permeation layer polymer.

[0018] In another embodiment, the permeation layer is a materialsuitable for transmitting electronic charge from an electrode to asolution overlaying the electrode. Materials contemplated forconstructing polymers used for the permeation layer may include, but arenot limited to, agarose, glyoxylagarose, acrylamide, methacrylamide,polyacrylamide, materials made from other synthetic monomers, hydrogels,and porous inorganic oxides created through a sol-gel process (Brinkeret al., Sol-Gel Science, Academic Press, San Diego, 1990).

[0019] Synthetic monomers used to make polymeric permeation layers mayinclude those selected from the group consisting of epoxides, alkenylmoieties including, but not limited to, substituted or unsubstituted α,β unsaturated carbonyls wherein the double bond is directly attached toa carbon which is double bonded to an oxygen and single bonded toanother oxygen, nitrogen, sulfur, halogen, or carbon; vinyl, wherein thedouble bond is singly bonded to an oxygen, nitrogen, halogen, phosphorusor sulfur; allyl, wherein the double bond is singly bonded to a carbonwhich is bonded to an oxygen, nitrogen, halogen, phosphorus or sulfur;homoallyl, wherein the double bond is singly bonded to a carbon which issingly bonded to another carbon which is then singly bonded to anoxygen, nitrogen, halogen, phosphorus or sulfur; and alkynyl moietieswherein a triple bond exists between two carbon atoms.

[0020] In another embodiment, the covalently attached permeation layeris kept from delaminating while the anode is charged with an electronicpotential above +1.29V/SCE and/or the cathode with a potential below−0.89 V/SCE. In a particularly preferred embodiment, the current flowbetween the electrodes has a density sufficient to induce the transportof molecules in the solution above the electrodes of the microchip. Suchdensity is preferably at least 0.04 nA/μm².

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The file of this patent contains at least one drawing executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee. The invention will be further described with reference tothe accompanying drawings in which:

[0022]FIG. 1 is a chemical structure schematic showing attachment of alinker moiety to the electrode surface.

[0023]FIG. 2 is a schematic diagram showing a process for covalentattachment of the permeation layer to the electrode. In the example ofthe figure, the electrode is treated with an argon plasma for 5 minutesat 250 mTorr (250W). This cleans the electrode, which has hydroxylfunctionalities at its surface. Linker is then attached to the electrodesuch as by vapor deposition for 5 minutes at room temperature followedby curing at 90° C. for 2 hours. This process leaves reactive moietiesthat can bond to the permeation layer. In the example of the figure, alinker having reactive amine groups is used wherein the amine moietiesare available for bonding to reactive moieties of the permeation layermatrix. The bonding between the linker and permeation layer reactivecenters can be accomplished using a Schiff base reaction.

[0024]FIG. 3A and B are confocal microscope photos of partial images ofindividual electrodes wherein the permeable membrane attached to theelectrode surface without use of a linker moiety is shown before (A) andafter (B) delamination.

[0025]FIG. 4A-D are confocal microscope photos of partial images ofindividual electrodes wherein the permeable membrane attached to theelectrode surface using AEAPS deposited by vapor is shown at variousdegrees of delamination.

[0026]FIG. 5A and B are confocal microscope photos of partial images of80 μm diameter Pt (A) and a PtSi (B) electrodes wherein the permeablemembrane was attached to the electrode surface using AEAPS. In FIG. 5A,the Pt electrode began to delaminate at the second direct currentimpulse of 500 nA (0.1 nA/μm²) for 2 min. In contrast, (FIG. 5B) thePtSi electrode showed no delamination after the second direct currentimpulse of 500 nA (0.1 nA/μm²).

[0027]FIGS. 6A and B, and 7A and B are confocal microscope photos ofpartial images of electrode arrays wherein the permeable membrane waseither deposited on a Pt electrode without chemical attachment (6A and7A) or was attached to a PtSi electrode surface using AEAPS (6B and 7B).The images show the levels of repeated biasing that result indelamination for Pt without covalent bonding of the permeation layer andPtSi microchips with covalent bonding.

[0028]FIGS. 8, 9, and 10 are confocal microscope photos of partialimages of a Pt electrode overlaid with agarose. In FIG. 8 the focalplane of the image is set at 3 μm above the electrode prior toelectronic biasing. This indicates the permeation layer surface is 3 μmabove the electrode as indicated by the beads being in focus. In FIG. 9an unchanged focal plane during electronic biasing is shown. In FIG. 10,a focal plane 4 μm above the electrode is shown indicating thatdelamination occurred causing the permeation layer to rise so that thebeads resting on top of the layer come into focus at a greater distancefrom the electrode.

[0029]FIG. 11 is a confocal microscope photo showing confirmation thatthe permeation layer of FIG. 10 delaminated as indicated by the presenceof concentric rings.

[0030]FIGS. 12 and 13 are confocal microscope photos of partial imagesof electrodes showing PtSi electrode with an acrylamide permeation layercovalently attached. FIG. 12 shows that the focal plane remainedunchanged after a two-minute bias at +2 μA (0.4 nA/μm²). FIG. 13confirms that no delamination occurred with this electrode.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] In the art of electronically addressable microchips used fortransporting charged molecules from one point in a solution to another,the transported molecules must be protected from direct contact with theelectrodes of the microchip and ions produced at the electrode when theelectrodes are biased to impart an electric field to the solution.Protection is provided by an insulating membrane, i.e., the permeationlayer, which also allows for the flow of charge from the electrode tothe solution without damaging the transported molecules. Typically, theinsulating membrane is a polymeric material such as agarose orcross-linked polyacrylamide. These materials are ideal in that they areporous and allow electrochemical products created at the electrode toescape to the overlying solution.

[0032] More specifically, such insulating membrane materials cancomprise, but are not limited to, agarose, glyoxylagarose, acrylamide,methacrylamide, polyacrylamide, materials made from other syntheticmonomers, and porous inorganic oxides created through a sol-gel process.Synthetic monomers used to make polymeric permeation layers may alsoinclude those selected from the group consisting of epoxides, alkenylmoieties including, but not limited to, substituted or unsubstituted α,β unsaturated carbonyls wherein the double bond is directly attached toa carbon which is double bonded to an oxygen and single bonded toanother oxygen, nitrogen, sulfur, halogen, or carbon; vinyl, wherein thedouble bond is singly bonded to an oxygen, nitrogen, halogen, phosphorusor sulfur; allyl, wherein the double bond is singly bonded to a carbonwhich is bonded to an oxygen, nitrogen, halogen, phosphorus or sulfur;homoallyl, wherein the double bond is singly bonded to a carbon which issingly bonded to another carbon which is then singly bonded to anoxygen, nitrogen, halogen, phosphorus or sulfur; and alkynyl moietieswherein a triple bond exists between two carbon atoms.

[0033] As described above, for optimal functionality of electronicallyaddressable microchips, it is important that the porous insulating layeror permeation layer remain in contact with the electrode in order toenhance uniformity and consistency of the electronic potential from onepad to the other. As shown in FIG. 1 the permeation layer may be linkedto the electrode by a linking moiety that has at least two reactivecenters. Linkers having suitable characteristics such as that shown inFIG. 1 are provided in Table I. TABLE I CHEMICAL TYPE FORMULA ACRYLATES:CH₂═CHCOOCH₂CH₂CH₂Si(OCH₃)₃ CH₂═CHCOOCH₂CH₂CH₂SiCl₃CH₂═CHCOOCH₂CH₂CH₂Si(CH₃)(OCH₃)₂ CH₂═CHCOOCH₂CH₂CH₂Si(CH₃)₂(OCH₃)CH₂═CHCOOCH₂CH₂CH₂Si(CH₃)Cl₂ CH₂═CHCOOCH₂CH(OH)CH₂NHCH₂CH₂CH₂Si(OC₂H₅)₃METHACRYLATES: CH₂═C(CH₃)COOCH₂CH₂CH₂Si(OCH₃)₃(MOTS)CH₂═C(CH₃)COOCH₂CH₂CH₂SiCl₃ CH₂═C(CH₃)COOCH₂CH₂CH₂Si(CH₃)(OCH₃)₂CH₂═C(CH₃)COOCH₂CH₂CH₂Si(CH₃)₂(OCH₃) CH₂═C(CH₃)COOCH₂CH₂CH₂Si(CH₃)Cl₂CH₂═C(CH₃)COOCH₂CH(OH)CH₂NHCH₂CH₂CH₂Si(OC₂H₅)₃ ACRYLAMIDES:CH₂═CHCONHCH₂CH₂CH₂Si(OC₂H₅)₃(AMPTS) CH₂═CHCONHCH₂CH₂CH₂SiCl₃CH₂═CHCONHCH₂CH₂CH₂Si(CH₃)(OCH₃)₂ CH₂═CHCONHCH₂CH₂CH₂Si(CH₃)₂(OCH₃)CH₂═CHCONHCH₂CH₂CH₂Si(CH₃)Cl₂CH₂═CHCONHCH₂CH(OH)CH₂NHCH₂CH₂CH₂Si(OC₂H₅)₃CH₂═CHCONHCH₂CH₂CONHCH₂CH₂CONHCH₂CH₂CH₂Si(OC₂H₅)₃ METHACRYLAMIDES:CH₂═C(CH₃)CONHCH₂CH₂CH₂Si(OCH₃)₃ CH₂═C(CH₃)CONHCH₂CH₂CH₂SiCl₃CH₂═C(CH₃)CONHCH₂CH₂CH₂Si(CH₃)(OCH₃)₂CH₂═C(CH₃)CONHCH₂CH₂CH₂Si(CH₃)₂(OCH₃) CH₂═C(CH₃)CONHCH₂CH₂CH₂Si(CH₃)Cl₂CH₂═C(CH₃)CONHCH₂CH(OH)CH₂NHCH₂CH₂CH₂Si(OC₂H₅)₃ ALLYL DERIVATIVES:CH₂═CHCH₂NHCH₂CH₂CH₂Si(OCH₃)₃ CH₂═CHCH₂SiH(OCH₃)₂ CH₂═CHCH₂Si(CH₃)₂ClCH₂═CHCH₂SiHCl₂ CH₂═CHCH₂Si(OCH₃)₃ AMINO DERIVATIVES:H₂NCH₂CH₂NHCH₂CH₂CH₂Si(OCH₃)₃(AEAPS)H₂NCH₂CH₂CH₂CH₂CH₂CH₂NHCH₂CH₂CH₂Si(OCH₃)₃(AHAPS)H₂NCH₂CH₂CH₂Si(OCH₃)₃(APS) H₂NCH₂CH₂CH₂Si(OC₂H₅)₃ EPOXY DERIVATIVES:

[0034] In a particularly preferred embodiment, microchips havingcovalent attachment chemistry of the current invention use linkersdenoted APS, AEAPS, AHAPS, MOTS, and AMPTS.

[0035]FIG. 2 shows a schematic of one embodiment wherein AEAPS is usedto bond the electrode to the permeation layer. In this example, the PtSielectrode microchip is first treated with an argon plasma for 5 minutesat 250 mTorr and 250 Watts. The chip is then treated with AEAPS by vapordeposition over 5 minutes at room temperature then cured onto the chipby heating for 2 hours at 90° C. This causes the linker to covalentlybind to the hydroxyl groups of the silicide moiety in the PtSielectrode. Once the linker is attached to the microchip, the permeationpolymer (for example glyoxylagarose) is overlaid onto the electrodesurface and treated in the presence of NaBH₃CN so that a Schiff basereaction and reduction can occur and cause the amine groups of the AEAPSlinker to bond to the aldehyde functionality available on the permeationpolymer (e.g., glyoxylagarose). Where polyacrylamide is employed as thepermeation layer polymer, a UV-initiated free radical polymerizationreaction can be conducted between the monomers which will make up thepermeation layer and the vinyl moieties present at the surface of MOTS-or AMPTS linker-derived electrodes, thereby synthesizing the permeationlayer and covalently anchoring it to the electrode in a single step.

[0036] Examples are provided below showing various delaminationthreasholds after attachment of the permeation layer using variouslinkers and attachment reaction conditions.

EXAMPLE 1

[0037] Agarose permeation layer matrix was attached to a PtSi electrodemicrochip following deposition of either APS or AEAPS by one of twomethodologies.

[0038] APS and AEAPS were deposited by exposure of the chip to a 0.1 wt% silane/dry MeOH solution for 1 hour at room temperature. The chipswere rinsed in EtOH and cured at 90° C. for 1 hour. In parallelexperiments, APS and AEAPS linkers were deposited onto microchips by avapor of neat silane in humid atmosphere for 5 min. at room temperaturefollowed by a two hour cure at 90° C.

[0039] After the agarose permeation layer was attached, the microchipswere subjected to electronic assays wherein the electrodes were biasedwith three direct current (DC) impulses for 2 minutes each at 200, 500,700, and 1000 nAmps/80 μm pad (i.e., 0.04, 0.10, 0.14 and 0.20 nA/μm²)using a model 236 Source-Measure unit (Keithley Instruments Inc.,Cleveland, Ohio). Following the set of three DC impulses, the electrodeswere biased with a sequence of 150 negative pulses, each comprised of a0.1 sec. ON state at −0.2 nA/μm², followed by a 0.2 sec. OFF state at 0nA/μm². As shown in Table II, the attachment schemes using vapordeposition of the linkers provided protection from delamination up to DCimpulses of 700 nA for an 80 μm electrode (0.14 nA/μm²). TABLE II 200200 200 −1 500 500 500 −1 700 700 nA nA nA uA nA nA nA uA nA nA samp DC1DC2 DC3 AC DC1 DC2 DC3 AC DC1 DC2 A PtSi (no + + + + + + + + + +permlayer) B PtSi/perm + +/− +/− − − − − − layer(no linker) CPtSi/APS/perm + + +/− − − − layer(dry MeOH deposited)* DPtSi/AEAPS/ + + + + + +/− − − − − perm layer(dry MeOH deposited)* EPtSi/APS/perm + + + + + +/− layer(vapor deposited)* FPtSi/AEAPS/ + + + + + +/− perm layer(vapor deposited)*

[0040] As shown in FIGS. 3 and 4, delamination will occur at low levelsof DC (200 nA (0.04 nA/μm²) after second DC pulse) where no covalentlinker attachment is used to anneal the permeation layer to theelectrode (FIGS. 3A and B). Conversely, where AEAPS is used that hasbeen applied to the electrode using vapor deposition, the delaminationdoes not appear until the electrode has been exposed to the second DCpulse at 700 nA (0.14 nA/μm²) (delamination extended to 25% of the padarea at 3 min. past shut-off) with complete delamination by 2 minutespast third DC shut-off (FIGS. 4A-D).

EXAMPLE 2

[0041] In this example, delamination of the permeation layer from theelectrode was tested using a multilayer permeation layer wherein thelayers were applied using spin coating techniques then reacted to causethe linking moieties to covalently bond the layers together and to theelectrode.

[0042] Specifically, microchips having PtSi electrodes were cleaned withoxygen plasma for 10 minutes followed by argon plasma for 10 minutes.AEAPS was then vapor-deposited for 5 minutes followed by curing at 90°C. under vacuum. Subsequently, a first layer solution comprising 2.5%glyoxylagarose solution (NuFix) which had been stirred for 10 minutes atroom temperature then boiled 7 minutes followed by filtering at 1.2 μminto the ASC device reservoir at 65° C., was spin-deposited onto themicrochips with an automatic spin-coating device (ASC). Followingdeposition of the first layer, a second layer, comprising streptavidin(Scripps Laboratory, San Diego) at 5 mg/ml in 10 mM sodium phosphate,250 mM NaCl (pH 7.2) which was filtered at 0.2 μm into the ASC reservoirand maintained at room temperature, was deposited similarly. The bottomlayer was spin-coated at either 1500 or 2500 rpm, while the top layerwas spin-coated at 5,000 rpm. The reaction for the reduction of theSchiff bases generated between streptavidin and glyoxylagarose, andbetween the AEAPS surface and glyoxylagarose was carried out by treatingthe coated microchip with 0.2 M NaBH₃CN 0.1 M sodium phosphate (pH 7.4)for 1 hr. at room temperature. Capping of the unreacted sites wasperformed by application of 0.1 M Gly/0.1 M NaBH₃CN, 0.1 M sodiumphosphate (pH 7.4) to the chip for 30 minutes at room temperature.Finally, the treated microchip was exhaustively rinsed and soaked indeionized water for 30 minutes and then air dried overnight at roomtemperature.

[0043] As shown in Table III below, the thickness of the doublepermeation layer was examined where the substrate contained either plainplatinum electrodes or PtSi electrodes using two different rotationalspeeds for the bottom layer deposition. The results indicate thatspin-coating results in deposition of permeation layers of variablethicknesses. TABLE III Bottom layer spun at 1.5K Bottom layer spun at2.5K rpm, bilayer thickness in rpm, bilayer thickness in Microchip typenanometers nanometers Pt/AEAPS/ 587 ± 4 465 ± 4 agarose 668 ± 4 465 ± 4668 ± 3 — PtSi/AEAPS/  744 ± 17 511 ± 4 agarose 685 ± 1 620 ± 5  494 ±90

[0044] The chips as fabricated in this example, were tested forresistance to delamination. For the platinum electrode microchips, 9electrode pads were individually addressed from two separate chips in 50mM fresh Histidine buffer. These pads showed consistent delaminationpast the second two-minute direct current pulse of 500 nA/80 μm pad (0.1nA/μm²) (FIG. 5A). In contrast, 6 pads were individually addressed from2 of the PtSi microchips under the same conditions. These PtSi pads hadno delamination up to several μA/pad (FIG. 5B). Thus, the PtSi electrodeusing the AEAPS attachment linker provided protection from delamination.

EXAMPLE 3

[0045] In this example, data is presented showing that the covalentattachment method of the invention using PtSi electrodes, agarose andaminopropylsilanes also protects against delamination of the permeationlayer under alternating current conditions. Here, Pt and PtSi microchipsbonded to the permeation layer with AEAPS were tested using two pulsedbiasing protocols.

[0046] Both protocols were carried out using 50 mM L-Histidine buffer.Specifically, in protocol A, the microchips were biased at +800 nA/pad(0.16 nA/μm²) for 38 milliseconds (ms), −800 nA/pad for 25 ms, cycledfor a total of 25 seconds using 3 pads each pulse. In protocol B, themicrochips were biased at +1.6 μA/pad (0.32 nA/μm²) for 19 ms, −1.6μA/pad for 12 ms, and cycled for a total of 14 seconds each on 3 padsaddressed simultaneously. Images were taken using an INM 100 confocalmicroscope (Leica).

[0047]FIG. 6A shows Pt chips that were biased using protocol A, followedby 0, 4, 8, 12 or 16 repeats of protocol B. The images show thatdelamination begins after 8 repeats of protocol B. In contrast, the PtSichips (FIG. 6B) showed delamination to a much less extent at the 8^(th)biasing. In order to more accurately define the delamination threshold,the chips were assayed with smaller stringency increments using biasingrepeats of 2, 4, 6, and 8 times. On Pt electrodes, delamination began tooccur at bias repeat number 6 (FIG. 7A). In contrast, the PtSi chipshowed less delamination effect at the same level of electrodynamicstress (FIG. 7B).

[0048] The overall results indicate that damage begins to occur duringthe sixth application of the above protocol B and that the delaminationincreased with increasing cycle repeats. This delamination effect wasless prominent in the PtSi chips.

EXAMPLE 4

[0049] In this example, methacryloylsilanes are employed as linkers forattaching synthetic permeation layers such as acrylamide-based hydrogelsto Pt and PtSi chips. Additionally, the integrity of the permeationlayer was examined using a technique wherein glass beads are applied tothe surface of the permeation layer as a reference upon which theconfocal microscope can focus. This enables permeation layer thicknessdetermination and facilitates the monitoring of permeation layerdistortions due to such things as delamination.

[0050]FIG. 8 shows a Pt microchip having an agarose permeation layerwherein the thickness of the layer before electronic biasing wasdetermined to be 3.0±0.5 μm. The figure shows the focal point at theposition of the beads above the electrode. Thus, the underlyingelectrode is slightly out of focus. FIG. 9, the same electrode during abias at +200 nA (0.04 nA/μm²) with direct current without observabledistortion of the permeation layer. The beads migrate to the electrodedue to the positive bias. Following this two-minute bias, the impulsewas terminated and the electrode observed for changes in its appearance.As seen in FIG. 10, the beads resting over the center of the electrodemoved to a location 4.0±0.5 μm above the electrode based on the verticalshift required to bring said beads back into the focal plane. Thus, thepermeation layer underwent a 1 μm expansion. As shown in FIG. 11, thisexpansion appears to be related to the delamination of the permeationlayer from the electrode (microdelamination) as indicated by thepresence of concentric rings visible at the edges of the electrode pad.Additionally, in other experiments, not shown, we have observedpermeation layer thickness distortions from 2 to 6 μm occurring withdelamination.

[0051] In another experiment, acrylamide-based hydrogel permeationlayers anchored to PtSi electrodes via the MOTS linker were exposed to a+200 nA (0.04 nA/μm²) bias for 2 minutes and examined for delamination.FIG. 12 shows beads resting atop the permeation layer 6 μm above theelectrode surface. The beads remained at the same position above theelectrode after bias shut-off, indicating that no distortion of thepermeation layer occurred. FIG. 13 shows the same pad with the focalpoint positioned at the electrode. No delamination ringlets wereobserved. When the electrodynamic stress was increased to +5 μA (1nA/μm²) for 2 mins., the permeation layer was observed to distort suchthat the layer seemed to swell. However, no delamination from theelectrode was observed. The results of the above experiments are shownin Table IV. TABLE IV bias conditions initial post (current dry wetaddress integrity of electrode/ chip type densities thickness thicknessdistortion permeation layer bond Pt/agarose 200nA, 2 min 0.80 ± 0.01 3.0± 0.5 4.0 ± 0.5 delamination & (0.04 nA/μm²) distortion 200nA, 2 min0.80 ± 0.01 3.0 ± 0.5 6.0 ± 0.5 delamination & (0.04 nA/μm²) distortionPt/poly 200nA, 2 min 2.0 ± 0.1 5.0 ± 0.5 9.0 ± 0.5 delamination &acrylamide (0.04 nA/μm²) distortion 200nA, 2 min 1.9 ± 0.1 5.0 ± 0.5 9.0± 0.5 delamination & (0.04 nA/μm²) distortion PtSi/poly 200nA, 2 min 2.0± 0.1 5.0 ± 0.5 5.0 ± 0.5 intact acrylamide (0.04 nA/μm²) 500nA, 1 min2.0 ± 0.1 6.0 ± 0.5 6.0 ± 0.5 intact (0.1 nA/μm²) 1 uA, 2 min 2.0 ± 0.16.0 ± 0.5 6.0 ± 0.5 intact (0.2 nA/μm²) 2 uA,2 min 2.0 ± 0.1 6.0 ± 0.56.0 ± 0.5 intact (0.4 nA/μm²) 5 uA, 2 min 2.0 ± 0.1 6.0 ± 0.5 12.0 ±0.5  distortion without (1 nA/μm²) delamination

[0052] Given that these results show that current densities in the rangeof 1 nA/μm² are useful in the operation of microchips having bondingchemistry resistant to delamination, we further contemplate that currentdensities in the range of at least 10 nA/μm² may be used with microchipshaving permeation layers which are bound to the electrodes using thebonding chemistry of the present invention without delamination.

[0053] Modifications and other embodiments of the invention will beapparent to those skilled in the art to which this invention relateshaving the benefit of the foregoing teachings, descriptions, andassociated drawings. The present invention is therefore not to belimited to the specific embodiments disclosed but is to includemodifications and other embodiments which are within the scope of theappended claims. All references are herein incorporated by reference.

We claim:
 1. An electronically addressable microchip comprising: a. atleast one electrode; b. a permeation layer; and c. linker moietiesconnecting said at least one electrode to said permeation layer, whereinsaid linker moieties are connected to said electrode and permeationlayer by covalent bonds, said covalent bonds capable of withstanding acurrent density of at least 0.04 nA/μm².
 2. An electronicallyaddressable microchip according to claim 1 wherein said permeation layeris selected from the group consisting of a polymer, a hydrogel, a porousinorganic oxides created through a sol-gel process, agarose,glyoxylagarose, and polymers synthesized from any of acrylamide,methacrylamide, or a synthetic monomer.
 3. An electronically addressablemicrochip according to claim 1 wherein said electrode is selected fromthe group consisting of platinum silicide (PtSi), tungsten silicide(WTi), titanium silicide (TiSi), gold silicide (AuSi), platinum/titanium(PtTi), gold /titanium (AuTi), poly(phenylene vinylene), polythiophene,and polyaniline.
 4. An electronically addressable microchip according toclaim 1 wherein said linker is has the formula

wherein X is selected from the group consisting of acrylate,methacrylate, acrylamide, methacrylamide, allyl, vinyl, acetyl, amine,substituted amine, epoxy and thiol; SPACER is selected from the groupconsisting of alkyl, aryl, mono- or polyalkoxy, ethyleneglycol,polyethyleneglycol, mono- or polyalkylamine, mono- or polyamide,thioether derivatives, and mono- or polydisulfides; A and B are selectedfrom the group consisting of Oxygen-R, Cl, Br, and an X-SPACER moiety,or any combination thereof, wherein R is H, alkyl, methyl, ethyl,propyl, isopropyl, and branched or linear alkyl of 4 to 10 carbon atoms,and C is a hydrolyzable moiety selected from the group consisting ofOxygen-R, Cl, and Br, wherein R is H, branched alkyl, methyl, ethyl,propyl, isopropyl, and branched or linear alkyl of 4 to 10 carbon atoms.5. An electronically addressable microchip according to claim 4 whereinsaid linker is selected from the group consisting of APS, AEAPS, AHAPS,MOTS, and AMPTS.
 6. A method of covalently attaching a permeation layerhaving reactive moieties to a metal/silicide, metal/metal or an organicelectrode of an electronically addressable microchip comprising: a.contacting said electrode with a linker molecule to form a product of anelectrode layered with said linker; b. contacting said product of (a)with a permeation layer matrix; and c. subjecting said product of (a)and matrix to a chemical reaction wherein a result of steps (a), (b) and(c) is covalent bonding between a first reactive moiety of said linkerand said electrode and a second reactive moiety of said linker and saidpermeation layer matrix, said covalent bonding capable of withstanding acurrent density of at least 0.04 nA/μm².
 7. A method according to claim6 wherein said metal/silicide electrode is selected from the groupconsisting of platinum silicide (PtSi), tungsten silicide (WTi),titanium silicide (TiSi), and gold silicide (AuSi).
 8. A methodaccording to claim 6 wherein said metal/metal electrode is selected fromthe group consisting of platinum/titanium (PtTi) and gold /titanium(AuTi).
 9. A method according to claim 6 wherein said organic electrodeis selected from the group consisting of poly(phenylene vinylene),polythiophene, and polyaniline.
 10. A method according to claim 6wherein said linker has the formula

wherein X is selected from the group consisting of acrylate,methacrylate, acrylamide, methacrylamide, allyl, vinyl, acetyl, amine,substituted amine, epoxy and thiol; SPACER is selected from the groupconsisting of alkyl, aryl, mono- or polyalkoxy, ethyleneglycol,polyethyleneglycol, mono- or polyalkylamine, mono- or polyamide,thioether derivatives, and mono- or polydisulfides; A and B are selectedfrom the group consisting of Oxygen-R, Cl, Br, and an X-SPACER moiety,or any combination thereof, wherein R is H, alkyl, methyl, ethyl,propyl, isopropyl, and branched or linear alkyl of 4 to 10 carbon atoms,and C is a hydrolyzable moiety selected from the group consisting ofOxygen-R, Cl, and Br, wherein R is H, branched alkyl, methyl, ethyl,propyl, isopropyl, and branched or linear alkyl of 4 to 10 carbon atoms.11. A method according to claim 10 wherein said linker is selected fromthe group consisting of APS, AEAPS, AHAPS, MOTS, and AMPTS.
 12. A methodaccording to claim 6 wherein said permeation layer is selected from thegroup consisting of a polymer, a hydrogel, a porous inorganic oxidescreated through a sol-gel process, agarose, glyoxylagarose, and polymerssynthesized from acrylamide, methacrylamide, polyacrylamide, or asynthetic monomer.
 13. A method according to claim 6 wherein saidbonding between said linker and permeation layer results from a Schiffbase reaction.
 14. A method according to claim 6 wherein said secondreactive moiety of said linker comprises an amine group.